The adult mouse incisor is a continuously growing organ that generates all necessary cell types from two distinct populations of epithelial and mesenchymal stem cells. The mouse incisor is therefore an excellent model to study the molecular regulation of adult organ regeneration. Recent work using mouse genetic models has uncovered a number of the molecular mechanisms regulating the epithelial stem cells. However, despite the advances in knowledge about the epithelium, the molecular regulation of the mesenchyme during incisor regeneration is still poorly understood. The polycomb repressive complex 1 (PRC1) epigentically modifies histone H2A through ubiquitination of lysine 119. Research in other contexts has shown that PRC1 is required in quiescent stem cells to maintain repression of genes related to differentiation. BMI1 polycomb ring finger oncogene (Bmi1) is a core PRC1 complex member that coordinates histone ubiquitination. Canonical PRC1 complexes bind to sites that have been previously methylated by the PRC2 complex, whereas non-canonical PRC1 is recruited to histones independently. Identifying the targets of BMI1 epigenetic modification will be crucial for understanding the molecular pathways required during incisor mesenchymal regeneration. While Bmi1 has been observed in discrete populations of mesenchyme, the precise expression pattern, molecular function/targets are still unknown. We hypothesize that epigenetic modification mediated by Bmi1 regulates expression of genes that will drive distinct incisor mesenchymal stem cell populations towards the appropriate committed fate. Preliminary evidence shows Bmi1 expression in distinct regions where putative slow cycling mesenchymal stem cells reside. The characterization of Bmi1-positive cells will be addressed in Aim 1 using immunostaining and lineage tracing techniques. Bmi1 null mice (Bmi1-/-) have incisors with dramatically reduced dentin, the tissue derived from mesenchymal stem cells. The molecular requirement and function of Bmi1 in mesenchymal stem cells will be examined in Aim 2 by means of ChIP-Seq. In different contexts, BMI1 has been reported to participate with both canonical and non-canonical PRC1 complexes as well independent of PRC1 entirely. The mechanism of BMI1 epigenetic modification in the incisor mesenchyme will be explored in Aim 3 using Co-IP/mass spectrometry. Stem-cell based strategies are a promising approach for regenerative medicine. However, a prerequisite to development of clinical therapies is a thorough understanding of the molecular mechanisms that regulate the ability of stem cells to contribute to organ renewal. Therefore, this work is significant, as it wil uncover the molecular targets and pathways that participate in renewal or differentiation during tooth regeneration.